Conventional wide field microscopy is based on formation of a high-magnification image of an illuminated sample using conventional microscope optics. In contrast, confocal microscopy is based upon the selective illumination of a small part of the sample, referred to as a target region, and on the selective collection of light emitted from the target region. Image formation is accomplished by scanning the position of the target region within the sample. Typically, the sample is illuminated with an illumination beam which is brought to a diffraction-limited (or nearly so) focus within the sample. Light emitted by the part of the sample within the focal region of the illumination beam is selectively collected and detected.
It is helpful to define an observation beam as being the beam that would be present if the optical detector in the above selective collection and detection arrangement were replaced by an optical source. Parts of the sample outside the observation beam are generally “not seen” by the detector. Thus the overlap of the illumination beam and observation beam defines the target region. Since it is generally desirable to decrease the size of the target region as much as possible, the illumination beam and observation beam are typically both brought to a small diffraction-limited focus (e.g., using a high numerical aperture (NA) lens having low aberration). Furthermore, the focal regions of the illumination beam and observation beam typically overlap (i.e., the two beams are typically confocal).
In the earliest confocal microscopes, the illumination beam and observation beams are collinear. In fact, frequently the same optical elements define the observation and illumination beams, and the observed signal is separated from the illumination light with a beamsplitter or directional coupler. When a beam is brought to a focus, the resulting focal region typically has an axial dimension several times larger than its transverse dimensions, especially if the focusing numerical aperture is less than 0.5. Here the axial direction is along the beam axis and the transverse directions are perpendicular to the beam axis. Thus, collinear illumination and observation beams typically provide a generally “cigar shaped” target region, having an axial dimension several times larger than its transverse dimensions.
More recently, for example in U.S. Pat. No. 5,973,828, non-collinear illumination and observation beams have been employed. Since the two beams intersect at an angle, the resulting target region is smaller than it would be for collinear beams. In particular, the target region can be roughly spherical and can have a radius on the order of the transverse beam dimensions.
A further variation on non-collinear illumination and observation beams is considered in U.S. Pat. No. 6,369,928, where two non-collinear illumination beams are supplied to the sample. In this arrangement, the illumination beam optics can conveniently define non-collinear observation beams (e.g., illumination optics 1 defines observation beam 2 and vice versa). Alternatively, light emitted from a sample region where the two illumination beams overlap can be selectively collected by optics other than the illumination beam optics.
However, the selective collection of light from such an arrangement poses a number of practical difficulties, especially when fluorescence radiation is of interest. For example, it is generally desirable to collect as much light as possible, and light collection increases as the lens NA increases because light from the sample is usually emitted in all directions. But, aberration of a simple lens also increases as lens NA increases, which usually obliges a lens designer to make a critical trade between resolution, complexity, working distance, and light collection efficiency. Thus high resolution microscope objectives can provide high NA, but the resulting designs are complex and bulky, and tend to provide a short working distance. This trade is applicable to both fluorescence microscopy and reflection/scattering microscopy. Furthermore, fluorescence is often emitted over a broad range of wavelengths, thus fluorescence confocal microscopes usually must further include chromatic aberration in the above trade, which greatly complicates their design. Finally, some applications, such as in vivo imaging require long working distances which are difficult to provide with high NA lenses.
Accordingly, it would be an advance in the art to provide a dual-illumination beam confocal microscope having a less critical trade between light collection, chromatic aberration, complexity, working distance, and resolution.